The Epstein Barr virus (EBV) in the human herpes virus that causes mononucleosis and is associated with some forms of cancer. After the initial mononucleosis infection, EBV persists in a latent state throughout the life of its host in lymphocytes as a 172kb duplex circular episome at a low copy number and only divides when the cellular chromosomes divide. Hence, the EBV latent origin of replication (oriP) is tightly regulated to coexist with the cell it lives in rather than to destroy it. The regulated replication of EBV may be a model for regulation of mammalian chromosome replication. With this hope we have overproduced and purified the viral encoded oriP binding protein, EBNA1, in milligram quantities using the baculovirus expression system. The project aim is to understand in molecular detail how EBNA1 regulates EBV replication. The oriP sequence is 1.8kb and is composed of two essential regions, an array of 21 tandem copies of a 30bp sequence and a dyad symmetry region that contains 4 of the 30bp sequences. EBNA1, the only viral encoded protein needed for latent replication, binds 18bp of this repetitive 30bp sequence in oriP. The first set of experiments proposed herein utilize [35S]-EBNA1, gel filtration analysis and DNA protection techniques to determine the stoichiometry, order and cooperativity of site binding by EBNA1 in each of the two elements of oriP and whether one oriP element influences the binding of EBNA1 to the other element. The activity of EBNA1 on oriP will be examined including ATPase and helicase assays and topological assays designed to detect wrapping bending or localized melting of oriP by EBNA1 or formation of a loop between the tow elements of oriP. Since EBNA1 is a phosphoprotein, its replication activity may be regulated by kinases or phosphatases. Nonphosphorylated EBNA1 (purified from the E. coli overproducer) and influence of phosphorylation state on EBNA1 action. Cellular factors that interact with sites in oriP or bind directly to EBNA1. The ultimate assay for purification of cellular initiation factors, an in vitro EBV replication system, will be attempted by supplementing EBNA1 and oriP plasmid DNA with cell extracts. The overall goal for this project in the long term is to use the intelligence (and cellular factors) gained from the EBV studies as a basis for future studies on the molecular mechanism(s) of the initiation and regulation of cellular chromosome replication.